Eosinophilic gastrointestinal disorders (EGID) (i.e. eosinophilic esophagitis [EE]) are being increasingly recognized over the last decade yet they have been greatly understudied compared with other allergic diseases. Over the past 9 years, we have been funded by the NIAID to study the regulation and role of murine GI eosinophils at baseline and in allergic inflammatory states. In the first grant cycle, we identified that eotaxin- 1 provides an essential signal for regulating the baseline and allergen-induced homing of eosinophils into the GI tract. Furthermore, we established a mechanistic link between the development of eosinophil-associated inflammation in the respiratory tract and the esophagus following specific aeroallergen or cytokine delivery to the lung. We identified a key role for IL-5 in eliciting esophageal eosinophilia and this provided the impetus for the current clinical trials now testing the role of humanized anti-IL-5 for the treatment of EE. In the second cycle of this grant, we published >30 papers, and extended these findings by identifying a key role for the paired Ig receptor (PIR)-B in negatively regulating GI eosinophil trafficking. In particular, we showed PIR-B to be expressed by murine eosinophils, to directly inhibit eotaxin-induced responses in eosinophils in vitro and to negatively regulate GI eosinophil recruitment in vivo. We used our murine model of experimental EE to identify key roles for Th2 cell adaptive immunity and IL-13 in the pathogenesis of EE, and to validate our findings in human EE that the disease pathogenesis involved an eotaxin-receptor dependent mechanism. Furthermore, we focused on mechanisms of esophageal remodeling, identifying a key role for IL-5-driven eosinophils and the connective tissue molecule periostin. Finally, we established a link between GI eosinophils and the pro- inflammatory mediator resistin like molecule (Relm)-1 by demonstrating that intestinal eosinophils are (1) a chief source of Relm-1;(2) Relm-1 is overproduced in experimental EE;and (3) Relm-1 has eosinophil chemotactic activity. The objective of our renewal application is to further understand mechanisms that regulate GI eosinophils. Our central hypothesis is that GI eosinophil responses are mediated by Relm-1 and counter-regulated by PIR-B. In Aim I, we will examine the role of Relm-1 in GI eosinophil responses, including the detection of signaling cascades in-situ by multi-color immunofluorescence. In Aim II, we will examine the role of PIR-B in GI eosinophil responses and eosinophil activation pathways. Finally, in Aim III, we will examine PIR-B and Relm-1 interactive signaling and function in eosinophils. Collectively, the proposed aims are designed to uncover the mechanisms by which eosinophils traffic and function in the GI tract. We combine innovative experimental approaches to dissect critical pathogenic mechanisms involved in experimental Th2 associated allergic GI responses. These results will shed important insight into the potential utility of immune-based therapeutics (e.g. anti-cytokine agents) for the treatment of EGID. PUBLIC HEALTH RELEVANCE: Eosinophilic gastrointestinal disorders (EGID) are a recently emerging medical problem typically associated with food allergies. We have developed experimental systems designed to understand the role and regulation of GI eosinophils, the principal white blood cell involved in these disorders. The current renewal application seeks to uncover the involvement of two newly described molecules that are regulatory checkpoints for GI eosinophils, Relm-1 (a cytokine released and active in eosinophils) and PIR-B (an inhibitory receptor expressed by eosinophils). These experiments will provide better therapeutic entry points for this rapidly emerging unmet allergy-associated medical problem.